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Overexpression of repetitive elements is an emerging hallmark of human cancers1. Diverse repeats can mimic viruses by replicating within the cancer genome through retrotransposition, or presenting pathogen-associated molecular patterns (PAMPs) to the pattern recognition receptors (PRRs) of the innate immune system2–5. Yet, how specific repeats affect tumor evolution and shape the tumor immune microenvironment (TME) in a pro- or anti-tumorigenic manner remains poorly defined. Here, we integrate whole genome and total transcriptome data from a unique autopsy cohort of multiregional samples collected in pancreatic ductal adenocarcinoma (PDAC) patients, into a comprehensive evolutionary analysis. We find that more recently evolvedShortInterspersedNuclearElements (SINE), a family of retrotransposable repeats, are more likely to form immunostimulatory double-strand RNAs (dsRNAs). Consequently, younger SINEs are strongly co-regulated with RIG-I like receptor associated type-I interferon genes but anti-correlated with pro-tumorigenic macrophage infiltration. We discover that immunostimulatory SINE expression in tumors is regulated by eitherLongInterspersedNuclearElements 1 (LINE1/L1) mobility or ADAR1 activity in aTP53mutation dependent manner. Moreover, L1 retrotransposition activity tracks with tumor evolution and is associated withTP53mutation status. Altogether, our results suggest pancreatic tumors actively evolve to modulate immunogenic SINE stress and induce pro-tumorigenic inflammation. Our integrative, evolutionary analysis therefore illustrates, for the first time, how dark matter genomic repeats enable tumors to co-evolve with the TME by actively regulating viral mimicry to their selective advantage.

SUMMARYSomatic chromosomal deletions are prevalent in cancer, yet their functional contributions remain ill-defined. Among the most prominent of these events are deletions of chromosome 9p21.3, which disable a cell intrinsic barrier to tumorigenesis by eliminating the CDKN2A/B tumor suppressor genes. However, half of 9p21.3 deletions encompass a cluster of 16 type I interferons (IFNs) whose co-deletions have not been functionally characterized. To dissect how 9p21.3 and other genomic deletions impact cancer, we developed MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements), a genome engineering strategy that enables flexible modeling of megabase-sized deletions. Generation of 9p21.3-syntenic deletions in a mouse model of pancreatic cancer revealed that concomitant loss of Cdkn2a/b and the IFN cluster led to immune evasion and metastasis compared to Cdkn2a/b-only deletions. Mechanistically, IFN co-deletion disrupted type I IFN signaling, altered antigen-presenting cells, and facilitated escape from CD8+ T cell surveillance in a cell extrinsic manner requiring loss of interferon epsilon (Ifne). Our results establish co-deletions of the IFN cluster as a pervasive route to tumor immune evasion and metastasis, revealing how deletions can disable physically linked cell intrinsic and extrinsic tumor suppression. Our study establishes a framework to dissect the functions of genomic deletions in cancer and beyond.

Single nucleotide variants (SNVs) comprise the majority of cancer-associated genetic changes and can have diverse effects on protein function. Despite a comprehensive catalogue of SNVs across human cancers, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a murine system for temporal and regulatable in vivo cytosine base editing (iBE). The iBE mice show robust, doxycycline-dependent expression across a broad range of tissues with no evidence of DNA or RNA off-target effects. Transient iBE induction drives efficient creation of individual or multiple SNVs in intestinal, lung, and pancreatic organoids, while temporal iBE regulation allows controlled sequential genome editing. Moreover, in situ delivery of plasmid-based or synthetic sgRNAs to target tissues facilitates the simple and rapid generation of pre-clinical cancer models. Overall, iBE is a powerful in vivo platform to define and interrogate the genetic drivers of cancer.

Cancer-associated fibroblasts (CAFs) are a major cell type in the stroma of solid tumors and can exert both tumor-promoting and tumor-restraining functions. This functional heterogeneity is correlated with the existence of transcriptionally distinct subpopulations of CAFs. CAF heterogeneity is observed in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by a remarkably dense and hypoxic stroma that features tumor-restraining myofibroblastic CAFs (myCAFs) and tumor-supporting inflammatory CAFs (iCAFs). While CAF heterogeneity can be driven in part by tumor cell-produced cytokines, other determinants shaping CAF identity and function are largely unknown. In vivo, we found that iCAFs display a hypoxic gene expression and biochemical profile and are enriched in hypoxic regions of PDAC tumors. Hypoxia leads fibroblasts to acquire an inflammatory gene expression signature and synergizes with cancer cell-derived cytokines to promote an iCAF phenotype in a HIF-1α dependent fashion. Furthermore, we show that HIF-1α stabilization is sufficient to induce an iCAF phenotype in stromal cells introduced into PDAC organoid co-cultures and to promote PDAC tumor growth. These findings indicate hypoxia-induced HIF-1α as a regulator of CAF heterogeneity and promoter of tumor progression in PDAC.

The response to tumor-initiating inflammatory and genetic insults can vary amongst morphologically indistinguishable cells, suggesting yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we perform single-cell analyses on normal, inflamed, pre-malignant and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identify heterogeneous cell-states that are primed for diverse late-emerging neoplastic fates and link these to chromatin remodeling at cell-cell communication loci. Using a new inference approach, we reveal signaling gene modules and tissue-level crosstalk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that we validate by genetic perturbation in mice. Our results uncover a neoplasia-specific tissue remodeling program that may be exploited for pancreas cancer interception.One-Sentence SummarySingle-cell analysis reveals that enhanced epigenetic plasticity drives pro-neoplastic crosstalk in early pancreatic cancer.

T cell-activating immunotherapies that produce durable and even curative responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be an effective approach to activate not only T cell but also cytotoxic Natural Killer (NK) cell-mediated anti-tumor immunity. Here we found that the pancreas tumor microenvironment (TME) suppresses NK and T cell surveillance following therapy-induced senescence through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity stimulated the production of pro-inflammatory SASP chemokines CCL2 and CXCL9/10 that led to enhanced NK and T cell infiltration and tumor eradication in preclinical PDAC mouse models. EZH2 activity was also associated with suppression of SASP-associated inflammatory chemokines and cytotoxic lymphocyte immunity and reduced overall survival in a PDAC patient cohort. These results demonstrate that EZH2 mediates epigenetic repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to potentiate NK and T cell surveillance in PDAC to achieve immune-mediated tumor control.

Metastatic gastric carcinoma is a highly lethal cancer that responds poorly to conventional and molecularly targeted therapies. Despite its clinical relevance, the mechanisms underlying the behavior and therapeutic response of this disease are poorly understood owing, in part, to a paucity of tractable models that faithfully recapitulate different subtypes of the human disease. To close this gap, we developed methods to somatically introduce different oncogenic lesions directly into the stomach epithelium and show that genotypic configurations observed in patients produce metastatic gastric cancers that recapitulate the histological, molecular, and clinical features of all non-viral molecular subtypes of the human disease. Applying this platform to both wild-type and immune-deficient mice revealed previously unappreciated links between the genotype, organotropism and immune surveillance of metastatic cells that produced distinct patterns of metastasis that were mirrored in patients. Our results establish and credential a highly portable platform for producing autochthonous cancer models with flexible genotypes and host backgrounds, which can unravel mechanisms of gastric tumorigenesis or test new therapeutic concepts aimed at improving outcomes in gastric cancer patients.

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3/KMT2C) histone methyltransferase occur in a range of solid tumors and heterozygous deletions encompassing MLL3 occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Mll3 cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded genes, Ink4a and Arf. Conversely, elevated MLL3 expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Mll3 restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in MLL3 and CDKN2A display mutual exclusivity in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

Cellular senescence involves a stable cell cycle arrest coupled to a secretory program that, in some instances, stimulates the immune clearance of senescent cells. Using an immune competent tumor model in which senescence triggers CD8 T cell-mediated tumor rejection, we show that senescence also remodels cell surface proteome to alter how they sense environmental factors, as exemplified by Type II interferon gamma (IFN-γ). Compared to proliferating cells, senescent cells upregulate IFN-γ receptor, become hypersensitized to microenvironmental IFN-γ, and more robustly induce antigen presenting machinery -effects also recapitulated in human tumor cells treated with senescence-inducing drugs. Disruption of the IFN-γ sensing by senescent cells blunts their immune-mediated clearance without disabling their characteristic secretory program or immune cell recruitment. Our results demonstrate that senescent cells have an enhanced ability to both send and receive environmental signals, and imply that each process is required for their effective immune surveillance.SIGNIFICANCEOur work identifies a novel interplay between tissue remodeling and tissue sensing programs that can be engaged by senescence in advanced cancers to render tumor cells more visible to the adaptive immune system. This new facet of senescence establishes reciprocal heterotypic signaling interactions that can be induced therapeutically to enhance anti-tumor immunity.

We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly gRNA/library design in custom genomes. GuideScan2 analysis identified widespread confounding effects of low-specificity gRNAs in published CRISPR knockout, interference and activation screens and enabled construction of a ready-to-use gRNA library that reduced off-target effects in a novel gene essentiality screen. GuideScan2 also enabled the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome.

We describe a new mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation; the resulting RC tumors invaded inward from the mucosal surface and metastasized to distant organs. Histologically the tumors closely resemble human RC and mirror remodeling of the tumor microenvirnoment (TME) in response to radiation. This murine RC model thus recapitulates the pathogenesis of human RC, thereby fulfilling the need for a physiologically accurate model for preclinical efficacy studies.

The chromatin adaptor Menin interacts with oncogenic fusion proteins encoded byMLL1-rearrangements (MLL1-r), and small molecules that disrupt these associations are currently in clinical trials for the treatment of leukemia. By integrating chromatin-focused and genome-wide CRISPR screens with genetic, pharmacological, and biochemical approaches in mouse and human systems, we discovered a molecular switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes that dictates response to Menin-MLL inhibitors. We show that MLL1-Menin safeguards leukemia survival by impeding binding of the MLL3/4-UTX complex at a subset of target gene promoters. Disrupting the interaction between Menin and MLL1 leads to UTX-dependent transcriptional activation of a tumor suppressor gene-program that is crucial for a therapeutic response in murine and human leukemia. We establish the therapeutic relevance of this mechanism by showing that CDK4/6 inhibitors allow re-activation of this tumor-suppressor program in Menin-inhibitor insensitive leukemia cells, mitigating treatment resistance. The discovery of a molecular switch between MLL1-Menin and MLL3/4-UTX complexes on chromatin sheds light on novel functions of these evolutionary conserved epigenetic mediators and is particularly relevant to understand and target molecular pathways determining response and resistance in ongoing phase 1/2 clinical trials.

The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) – a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation ofMYCand its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.

Genome copy number is an important source of genetic variation in health and disease. In cancer, clinically actionable Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in terms of the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms. This represents a challenge for applications that require high read counts such as CNA inference. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that sequencing short DNA molecules reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for the sequencing of relatively short DNA molecules on nanopore devices with applications in research and medicine, that include but are not limited to, CNAs.

While the non-coding genome appears to play a role, the dynamic nature of noncoding alterations with respect to clonal progression of solid tumors remains unexplored. To address this gap in knowledge we performed multiregional whole genome sequencing and clonal analysis to elucidate the evolutionary dynamics of non-coding regions in pancreatic cancer relative to those of the coding genome. We find that the mutational burden of noncoding DNA is higher than coding DNA. However, when noncoding DNA was segregated into enhancer and non-enhancer regions, enhancers were more similar to coding DNA. Mutational signatures of noncoding and coding DNA further revealed the similar mutational spectra of enhancers to coding DNA whereas the mutational spectra of non-enhancer, noncoding DNA had an entirely different pattern. These findings shed light on the role of noncoding DNA in pancreatic cancer.

The cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity, but the mechanisms governing cell-of-origin–driven differences in therapeutic response have not been delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and highly express the transcription factor MECOM (EVI1) compared with leukemias derived from myeloid progenitors. Here, we compared leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for EVI1 in modulating p53 protein abundance and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 (LSD1) inhibitors in addition to classical genotoxic stresses. p53 loss of function in Evi1lo progenitor-derived leukemias induces resistance to LSD1 inhibition, and EVI1hi leukemias are sensitized to LSD1 inhibition by venetoclax. Our findings demonstrate a role for EVI1 in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in chemoresistant EVI1hi acute myeloid leukemia. Significance: We demonstrate that the cell of origin of leukemia initiation influences p53 activity and dictates therapeutic sensitivity to pharmacologic LSD1 inhibitors via the transcription factor EVI1. We show that drug resistance could be overcome in HSC-derived leukemias by combining LSD1 inhibition with venetoclax. See related commentary by Gu et al., p. 1445. This article is highlighted in the In This Issue feature, p. 1426

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to individual patients. We established a biorepository of 32 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and theirex vivoresponses to clinically relevant chemotherapy and radiation treatment correlate well with responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive rectal cancer followed by metastasis to lung and liver. Importantly, engrafted tumors closely reflected the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based,in vitroplatform coupled with endoluminal propagation in animals.

CRISPR base editing is a potentially powerful technology that enables the creation of genetic mutations with single base pair resolution. By re-engineering both DNA and protein sequences, we developed a collection of constitutive and inducible base editing vector systems that dramatically improve the ease and efficiency by which single nucleotide variants can be created. This new toolkit is effective in a wide range of model systems, and provides a means for efficientin vivosomatic base editing.

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-xL and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-xL, Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, we identified the nuclear factor-κB (NF-κB) subunit p65 as a major transcription factor that accumulates on chromatin of senescent cells. We found that NF-κB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-κB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-κB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. Our results demonstrate that NF-κB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-κB that contributes to the outcome of cancer therapy.

Deletions on chromosome 8p are common in human tumors, suggesting that one or more tumor suppressor genes reside in this region. Deleted in Liver Cancer 1 (DLC1) encodes a Rho-GTPase activating protein and is a candidate 8p tumor suppressor. We show that DLC1 knockdown cooperates with Myc to promote hepatocellular carcinoma in mice, and that reintroduction of wild-type DLC1 into hepatoma cells with low DLC1 levels suppresses tumor growth in situ. Cells with reduced DLC1 protein contain increased GTP-bound RhoA, and enforced expression a constitutively activated RhoA allele mimics DLC1 loss in promoting hepatocellular carcinogenesis. Conversely, down-regulation of RhoA selectively inhibits tumor growth of hepatoma cells with disabled DLC1. Our data validate DLC1 as a potent tumor suppressor gene and suggest that its loss creates a dependence on the RhoA pathway that may be targeted therapeutically.

The p53 tumor suppressor plays a key role in organismal aging. A cellular mechanism postulated to drive the aging process is cellular senescence, mediated in part by p53. Although senescent cells accumulate in elderly individuals, most studies have relied on correlating in vitro senescence assays with in vivo phenotypes of aging. Here, using two different mouse models in which the p53-related protein p63 is compromised, we demonstrate that cellular senescence and organismal aging are intimately linked and that these processes are mediated by p63 loss. We found that p63+/- mice have a shortened life span and display features of accelerated aging. Both germline and somatically induced p63 deficiency activates widespread cellular senescence with enhanced expression of senescent markers SA-β-gal, PML, and p16INK4a. Using an inducible tissue-specific p63 conditional model, we further show that p63 deficiency induces cellular senescence and causes accelerated aging phenotypes in the adult. Our results thus suggest a causative link between cellular senescence and aging in vivo, and demonstrate that p63 deficiency accelerates this process.

The N-terminal region of adenoviral E1A interacts with p400, a member of the SWI2/SNF2 chromatin remodeling family (Fuchs et al. 2001). E1A mutants defective in interacting with p400 cannot transform primary cells efficiently when coexpressed with activated Ras (Deleu et al. 2001; Fuchs et al. 2001; Seger et al. 2002), suggesting that p400/E1A complexes participate in the transformation process. p400 interacts with SV40 large T antigen (Lill et al. 1997b), c-Myc (Fuchs et al. 2001), and the tumor suppressor, p53 (Lill et al. 1997a). It is also a component of the human TIP60/NuA4 complex (Ikura et al. 2000; Fuchs et al. 2001; Cai et al. 2003; Doyon et al. 2004), which has specific histone H4/H2A acetyltransferase activity (Ikura et al. 2000; Doyon et al. 2004). ChIP (chromatin immunoprecipitation) analysis has detected p400 complexes at certain c-Myc and E2F-regulated promoters (Frank et al. 2003; Taubert et al. 2004). Their recruitment to such promoters correlated with an increase in histone acetylation in the surrounding chromatin and induction of transcription. These findings suggest that p400-containing complexes exert key chromatin-remodeling functions and participate in transcription regulation. In the present study, retroviral-mediated shRNA was used to target p400 complexes and to investigate the outcomes of this perturbation in human cells. Specifically, acute depletion of p400 induced premature senescence in multiple, untransformed human fibroblasts. p400 shRNA-induced SAHF and other premature senescence phenotypes were rescued by coexpression of shRNA for p53 or p21, but not pRb. p400-depleted cells revealed elevated p21 mRNA and protein expression. In keeping with these findings, p400 was detected at or near the p53-binding site in the p21 promoter. These data suggest that p400 complexes inhibit p53 → p21 expression, and that deregulated p21 expression leads to senescence.

Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.

Oncogenicrasprovokes a senescent-like arrest in human diploid fibroblasts involving the Rb and p53 tumor suppressor pathways. To further characterize this response, we compared gene expression patterns betweenras-arrested and quiescent IMR90 fibroblasts. One of the genes up-regulated duringras-induced arrest was promyelocytic leukemia (PML) protein, a potential tumor suppressor that encodes a component of nuclear structures known as promyelocytic oncogenic domains (PODs). PML levels increased during bothras-induced arrest and replicative senescence, leading to a dramatic increase in the size and number of PODs. ForcedPMLexpression was sufficient to promote premature senescence. Like oncogenicras, PMLincreased the levels of p16, hypophosphorylated Rb, phosphoserine-15 p53, and expression of p53 transcriptional targets. The fraction of Rb and p53 that colocalized with PML markedly increased duringras-induced arrest, and expression ofPMLalone forced p53 to the PODs. E1A abolishedPML-induced arrest and prevented PML induction and p53 phosphorylation in response to oncogenicras. These results imply that PML acts with Rb and p53 to promoteras-induced senescence and provide new insights into PML regulation and activity.

The p53 tumor suppressor activates either cell cycle arrest or apoptosis in response to cellular stress. Mouse embryo fibroblasts (MEFs) provide a powerful primary cell system to study both p53-dependent pathways. Specifically, in response to DNA damage, MEFs undergo p53-dependent G1 arrest, whereas MEFs expressing the adenovirus E1A oncoprotein undergo p53-dependent apoptosis. As the p53-dependent apoptosis pathway is not well understood, we sought to identify apoptosis-specific p53 target genes using a subtractive cloning strategy. Here, we describe the characterization of a gene identified in this screen, PERP, which is expressed in a p53-dependent manner and at high levels in apoptotic cells compared with G1-arrested cells. PERP induction is linked to p53-dependent apoptosis, including in response to E2F-1-driven hyperproliferation. Furthermore, analysis of the PERP promoter suggests that PERP is directly activated by p53. PERP shows sequence similarity to the PMP-22/gas3 tetraspan membrane protein implicated in hereditary human neuropathies such as Charcot–Marie–Tooth. Like PMP-22/gas3, PERP is a plasma membrane protein, and importantly, its expression causes cell death in fibroblasts. Taken together, these data suggest that PERP is a novel effector of p53-dependent apoptosis.

Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16INK4a tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 orINK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19ARF. The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19ARF restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19ARF functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19ARF and DNA damage pathways in inducing p53 may contribute to E1A’s ability to enhance radio- and chemosensitivity.

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.

The majority of human cancers acquire mutations that abrogate the p53 tumor suppressor network and, as a consequence, p53 is one of the most extensively studied proteins in cancer research. Because of its potent tumor suppressive activity, it is widely assumed that a molecular understanding of p53 action will produce fundamental insights into natural processes that limit tumorigenesis and may identify key molecular targets for therapeutic intervention. p53 functions largely as a transcription factor, and can trigger a variety of antiproliferative programs by activating or repressing key effector genes. Despite a significant body of literature detailing the biochemical and biological functions of p53, much remains to be elucidated. Indeed, the p53 network is as complex and enigmatic as it is relevant. It is the goal of this article, written 30 years after the discovery of p53, to present a concise review of the tumor suppressor role of the p53 network and to highlight the context-dependent nature of p53 target-gene functions.